Unique immune regulation by alternatively spliced interleukin-4

描述(由申请人提供)： 白介素4在调节免疫稳态和各种免疫介导性疾病中发挥核心作用，包括但不限于哮喘、过敏、结核病和自身免疫相关的间质性肺部疾病(硬皮病、类风湿性关节炎、多发性肌炎和皮肌炎)。我们最近描述了一种IL-4的剪接变体，即所谓的IL-442。IL-442的生物学特性似乎与IL-4有很大的不同，然而该变体的功能仍然知之甚少。我们发现，IL-442在包括哮喘在内的各种疾病中的表达水平显著增加，达到与IL-4相似或超过IL-4的水平。此外，我们最近发现，IL-442改变了人T细胞中许多基因的表达水平，并且IL-442诱导的表达谱变化不同于IL-4诱导的变化。与IL-4不同，IL-442在广泛的测试浓度范围内不会刺激STAT6的磷酸化。与IL-4类似，IL-442以时间和剂量依赖的方式刺激T细胞中JAK1、JAK3和TYK2的磷酸化。IL-442基因导入小鼠肺内可引起肺环境的致炎改变，其诱导的肺组织中肿瘤坏死因子-1、IL-11、干扰素-3、IL-12p40和单核细胞趋化蛋白-1的水平明显高于IL-4基因导入所诱导的水平。哮喘患者支气管肺泡灌洗液和外周血T细胞中IL-442的相对表达水平多数高于IL-4。我们还发现，IL-442有效地刺激人类T细胞产生MCP-1，更有效地(数百倍)以自分泌方式上调IL-442的产生。根据我们的初步数据，本研究的具体假设是，IL-442与T细胞上的特定细胞表面受体结合；激活不同于IL-4诱导的信号的特有的细胞内信号；以不同于IL-4诱导的方式改变基因表达，特别是刺激自身以自分泌方式产生；最终刺激大量促炎分子和Th1分子的产生。此外，假设IL-442在活体内会导致肺部显著的促炎变化，并与人类患者更严重的哮喘有关。在具体目标1中，将在细胞培养中研究IL-442对原代人类淋巴细胞的影响，以及在分子水平上对其产生的调节。在具体目标2中，将在正常小鼠和卵清蛋白致敏的哮喘小鼠模型中研究体内IL-442基因传递的效果。在具体目标3中，将研究IL-442与哮喘患者疾病严重程度的关系。这项研究与退伍军人医疗保健直接相关。许多由IL-4介导的疾病没有已知的治疗方法，在普通人群中广泛传播，特别是在退伍军人中。这些疾病是慢性的、严重的、使人衰弱的，甚至是致命的。这项研究的优势在于，它以机械学的方式解决了一个新的主题；它结合了细胞培养、实验动物和人类患者的机械学研究；它涉及一种与退伍军人中流行的许多疾病的机制有关的分子。这项研究有望产生丰富的关于IL-442作为潜在诊断标记物的新信息，以及未来对退伍军人多种疾病的治疗调整的重要靶点。 公共卫生相关性： 体内一种名为白介素4(IL-4)的分子的产生可能会增加，并推动肺、肝、皮肤和其他器官的疾病进程。所有这些依赖IL-4的慢性疾病都可能随着年龄的增长或军事暴露的结果而发展，从而使它们在退伍军人中流行。我们最近发现IL-4有两种形式，传统的IL-4和一种称为IL-4-Delta-2(IL-442)的形式。我们发现，这种另一种形式在患者体内过度产生，在人体细胞上的作用与传统的IL-4不同。这项拟议的研究将着眼于IL-442对从人类分离的细胞的影响，调查小鼠肺中过量的IL-442导致的疾病，并确定人类体内过量的IL-442如何与疾病严重程度相关。这一结果将为开发针对患者，特别是退伍军人的IL-442的新疗法奠定基础，目的是减轻或治愈退伍军人的疾病，从而改善退伍军人的健康。

DESCRIPTION (provided by applicant): Project Summary Interleukin (IL)-4 plays a central role in the regulation of immune homeostasis and in various immune-mediated diseases including but not limited to asthma, allergies, tuberculosis, and autoimmunity-associated interstitial lung disease (scleroderma, rheumatoid arthritis, poly- and dermatomyositis). We recently described a splice variant of IL-4, so-called IL-442. The biology of IL-442 appears to be very different from that of IL-4, however the functions of this variant are still poorly understood. We found that the expression levels of IL-442 are dramatically increased in various diseases, including in asthma, to levels that are similar to or exceed those of IL-4. Furthermore, we recently found that IL-442 changes expression levels of numerous genes in human T cells, and that the IL-442-induced changes in the expression profile differ from those induced by IL-4. Unlike IL- 4, IL-442 does not stimulate phosphorylation of STAT6 in a broad range of tested concentrations. Similar to IL- 4, IL-442 stimulates phosphorylation of Jak1, Jak3, and Tyk2 in T cells in a time- and dose-dependent fashion. Gene delivery of IL-442 to mouse lungs causes proinflammatory changes in pulmonary milieu, with the induced levels of TNF-1, IL-11, IFN-3, IL-12p40, and MCP-1 significantly exceeding those induced by gene delivery of IL-4. In bronchoalveolar lavage and blood T cells of patients with asthma, relative expression levels of IL-442 were in many cases higher than those of IL-4. We also discovered that IL-442 potently stimulates production of MCP-1 in human T cells and even more potently (by several hundred fold) upregulates IL-442 production in autocrine fashion. Based on our preliminary data, the Specific Hypothesis of this study is that IL-442 binds to a specific cell surface receptor on T cells; activates characteristic intracellular signaling that is different from signaling induced by IL-4; changes gene expression in a fashion different from that induced by IL-4, particularly stimulating its own production in autocrine fashion; and ultimately stimulates production of numerous proinflammatory and Th1 molecules. Furthermore, the hypothesis is that IL-442 causes significant proinflammatory changes in the lungs in vivo, and is associated with more severe asthma in human patients. In Specific Aim 1, the effects of IL-442 on primary human lymphocytes, as well the regulation of its production at the molecular level, will be investigated in cell culture. In Specific Aim 2, effects of IL-442 gene delivery in vivo will be investigated in normal mice as well as in an ovalbumin-sensitized mouse model of asthma. In Specific Aim 3, the association of IL-442 with disease severity in asthma patients will be studied. This study is directly relevant to Veterans Healthcare. Many of the IL-4-mediated diseases have no known cures and are widely spread in the general population and particularly in veterans. These diseases are chronic, severe, debilitating, and even deadly. The strengths of this investigation are that it addresses a novel topic in a mechanistic way; that it combines mechanistic studies in cell culture, in experimental animals, and in human patients; and that it deals with a molecule that is involved in the mechanisms of numerous diseases that are prevalent in veterans. This study is expected to generate a wealth of novel information on IL-442 as a potential diagnostic marker as well as important target for future therapeutic modulation of numerous diseases in veterans. PUBLIC HEALTH RELEVANCE: Production of a body molecule called interleukin-4 (IL-4) can be elevated and drive disease processes in the lungs, liver, skin, and other organs. All of these IL-4-dependent chronic diseases may develop with aging or as a consequence of military exposures, thus making them prevalent in the veteran population. We recently discovered that IL-4 is made in two forms, traditional IL-4 and a form called IL-4-delta-2 (IL-442). We discovered that this other form is excessively produced in patients and acts differently from the traditional IL-4 on the cells of the body. The proposed research will look at the effects of IL-442 on the cells isolated from humans, investigate the disease caused by excess IL-442 in mouse lungs, and determine how excess IL-442 in humans is associated with disease severity. The results will form the basis for development of new therapies that target IL-442 in patients, particularly in veterans, with the goal of attenuating or curing diseases in veterans, and thus improving veterans' health.